Small interfering RNA (siRNA) is a double-stranded RNA of 19 to 21 base pairs and is a major molecule responsible for RNA interference (RNAi) known as a potent and specific gene expression silencing phenomenon (Non Patent Literature 1). In 2001, Tuschl et al. reported RNAi in mammalian cells (Non Patent Literature 2). Since then, research and development have been actively conducted towards applications of siRNA to treatments of any kinds of diseases. In this regard, however, the preceding development is directed to sites such as eyes and respiratory organs, in each of which siRNA easily reaches the affected part, resulting in a limited application range. In the first place, it is known that siRNA is an unstable chemical species easily degradable under a physiological environment, and undergoes rapid renal excretion when being intravenously administered alone to mice (t½=several minutes). Thus, there is a strong demand for a drug delivery system (DDS) that improves the pharmacokinetics of siRNA, which is regarded as crucial for the practical use of siRNA.
However, a systemic DDS still remains at a laboratory level and is far from practical use. Even a system of stable nucleic acid lipid particles (SNALP) collaboratively developed by Alnylam and Protiva Biotherapeutics, both of which have published most pioneering accomplishments, is hardly applied to tissues other than the liver because of its passive uptake into the liver (Non Patent Literature 3). On the other hand, for only a problem of long-term retention in the bloodstream, a system of wrapped liposomes developed by Kyowa Hakko Kogyo Co., Ltd., though directed to a single-stranded DNA, succeeded in markedly improving retention (Non Patent Literature 4). Hence, the system is greatly expected to be applicable to siRNA as well in the future. However, a system using liposomes, which is too stable, often makes it difficult to release a drug in the system.
As described above, a technology that improves the pharmacokinetics of siRNA and allows the administration of siRNA in the bloodstream is extremely important in expanding applications of siRNA. It has hitherto been reported that, regarding plasmid DNA or antisense DNA, a nanometer-scale structure (i.e., polymer micelle) encapsulating the DNA, which is formed using a polyethylene glycol-polylysine block co-polymer (PEG-PLys), is useful (NonPatent Literatures 5 to 8). Such polymer micelle is formed by self-assembly through an electrostatic interaction between a polycation moiety (polylysine moiety in PEG-PLys) in the block co-polymer and a nucleic acid molecule (e.g., DNA) as a polyanion. The formed polymer micelle has a core-shell type structure in which a polyion complex moiety including the polycation and the polyanion serves as an inner core-like moiety and its surface layer is covered with polyethylene glycol (PEG). Therefore, the polymer micelle is expected to potentially avoid the mechanism of xenobiotic recognition and renal excretion.
The inventors of the present invention have made an attempt at forming a polymer micelle encapsulating siRNA using PEG-PLys as a measure for improving the pharmacokinetics of siRNA in view of the fact that the plasmid DNA or antisense DNA is an siRNA analogue. However, the polymer micelle actually obtained was low in structural stability, did not sufficiently have a desired core-shell type micelle structure, and was extremely poor in siRNA delivery capacity to cultured cancer cells. Therefore, it was extremely difficult to utilize PEG-PLys as a carrier for siRNA.
Further, a polyion complex (PIC) formed through an electrostatic interaction between anionic siRNA and a polycation is utilized in a DDS (Patent Literature 1 and Non Patent Literatures 9 to 11). However, siRNA hardly forms a stable micelle under a physiological condition mainly because of its small size (21 base pairs). Hence, the stabilization of a PIC in a biological environment is a current problem (Non Patent Literature 12). In order to form a stable PIC, preferred is a long double-stranded RNA (dsRNA) having a large number and high density of anionic charges. This is because there occurs a cumulative interaction with a polycation. However, a long dsRNA structure involves a problem in that it causes IFN-α responses via a plurality of passways including recognition by Toll-like receptor-3 (TLR3) and activation of protein kinase R (PKR) (Non Patent Literatures 13 to 15).
In addition, microRNA (miRNA) is also a small RNA like siRNA, is capable of silencing gene expression through the same mechanism as siRNA, and hence is expected to be utilized in a DDS like siRNA. However, a DDS using miRNA also involves the same problem as in the case of siRNA.
As described above, there is a strong demand for such means for delivering small RNAs (for example, siRNA and miRNA) as to be stable in a biological environment without causing any undesired immune response.